Culture, formulation, and delivery techniques of Valdensinia heterodoxa, and its use as a biological control agent of salal (Gaultheria shallon)

ABSTRACT

The invention disclosed relates to a naturally occurring fungus,  Valdensinia heterodoxa , and to its culture, formulation and delivery systems, as well as its use as a biocontrol agent for salal ( Gaultheria shallon  Pursh.).

BACKGROUND OF THE INVENTION

[0001] The present invention relates to the naturally occurring fungus,Valdensinia heterodoxa, and in particular to its use as a biocontrolagent for salal (Gaultheria shallon Pursh.).

[0002] Forest management has become increasingly intensive in order tomaximize forest productivity and sustainability. The past few decadeshave seen significant changes in forest management practices, especiallyin the area of site preparation, the use of chemical herbicides, anddevelopment of new forest harvesting systems. Competition fromnon-commercial or competing forest vegetation is a major problem atconifer regeneration sites following harvest in plantations. Thiscompetition results in conifer mortality, reduced growth, delays inharvesting time, increased costs related to forest management, anddecreases in annual allowable cut (Wall et al. 1992). Management ofcompeting forest vegetation can take various forms, including removal bymechanical or manual brushing and chemical herbicides. These methodshave distinct disadvantages such as non-target effects and publicconcerns about the negative impacts of using herbicides in pristineforest ecosystems. Hence, there is a growing need for alternativemanagement strategies for competing vegetation that are cost-effective,environmentally safe, economically feasible, and sustainable (Watson andWall 1995). One viable option is the use of naturally occurring plantpathogens as biological control agents which, if successful, areexpected to result in increased early conifer growth rate and a shorterrotation age of commercially valuable crop trees (Shamoun 2000).

[0003] Salal (Gaultheria shallon Pursh.), a perennial, ericaceous shrub,is a serious competitor with conifer seedlings in coastal BritishColumbia. Generally, it competes with trees for water and nutrients andremoval of salal leads to enhanced conifer growth (McDonald 1990). Salalis difficult to control with current mechanical methods due to itsextensive root system leading to quick reestablishment through layering,sprouting, and suckering (D'Anjou 1990). Chemical herbicides are oftenineffective since salal's thick and leathery leaves reduce herbicidetranslocation (D'Anjou 1990). Hence, salal can be considered a suitabletarget weed for biological control using fungal pathogens.

[0004] Numerous fungal species have been isolated from salal plants,including Mycosphaerella gaultheriae (Haeussler et al. 1990),Phyllosticta pyrolae Ellis et Everh. (Petrini et al. 1982), Phytophthoracinnamoni Rands (Lindermann & Zeitoun 1977), and Valdensinia heterodoxaPeyr. (Readhead 1974).

[0005] In 1999, a survey was conducted to collect and identify themycobiota associated with salal from various locations on VancouverIsland (Shamoun et al. 2000).

[0006] Fungal pathogens isolated from diseased leaf and stem tissue weresubsequently assessed for their virulence on salal. From the testedfungi, Valdensia heterodoxa (PFC 3027) caused substantial leaf damage onboth detached leaves and intact salal plants (Vogelgsang et al. 2001).

SUMMARY OF THE INVENTION

[0007] According to an embodiment of the present invention, abiologically pure isolate of the naturally occurring fungus, Valdensiniaheterodoxa, having all of the identifying characteristics of IDACDeposit Accession no. IDA 180402, is provided.

[0008] According to a further embodiment of the invention, a herbicidalcomposition containing as active ingredient, Valdensinia heterodoxa,having all of the identifying characteristics of IDAC Deposit Accessionno. IDA 180402, is provided.

[0009] According to yet another embodiment of the present invention, aherbicidal composition for controlling salal (Gaultheria shallon Pursh)is also provided, the composition comprising as active ingredient, aneffective amount of a culture of V. heterodoxa on an agriculturally andenvironmentally acceptable solid growth substrate capable of supportinggrowth of the fungus, containing a cereal grain, e.g., oatmeal.

[0010] According to yet another embodiment of the present invention, amethod for controlling salal (Gaultheria shallon Pursh.), is alsoprovided, the method comprising applying to a salal plant or to a salalplant locus, an effective amount of a herbicidal composition containingas active ingredient, a biologically pure isolate of the fungusValdensinia heterodoxa having all of the identifying characteristics ofIDAC Deposit Accession no. IDA 180402.

[0011] According to another embodiment of the present invention, amethod is provided for isolating Valdensinia heterodoxa from nature inbiologically pure form.

[0012] According to yet another aspect of the invention, salal planttisue e.g. leaves and stems colonized by V. heterodoxa is used as aninoculum delivery mechanism to control salal.

[0013] According to yet another embodiment of the present invention, aninoculation of salal plant tissue, e.g., leaf pieces, with mycelium ofV. heterodoxa produced from cereal grain, e.g., oatmeal, containinggrowth media e.g. agar.

[0014] We have also found that using additional solid substratesinfected with the fungus such as: 1) alder saw dust; 2) fir saw-dust; 3)vermiculite powder; 4) rice grain; 5) rolled oats; and 6) whole oats.Again, the results showed that the most effective solid substrate forformulation and conidia production and discharge was on salal leaves andstems. There was also production and discharge of conidia on alder sawdust and fir-saw dust, but was not significant compared to salal leavesand stems.

[0015] Further, we have found that a medium containing rolled oats asthe only carbohydrate source (e.g., water agar containing a few rolledoats on the surface) has triggered the sporulation of Valdensiniaheterodoxa.

[0016] According to yet another aspect of our invention, we have foundto advantage that innoculation of plant materials, such as leaves andstems as a delivery mechanism. Typically, in the prior art, non-hostmaterials are used such as cereal grains, alginate pellets andvermiculite.

[0017] As already mentioned above, in the culturing method itself, theuse of oatmeal in the medium, lower incubation temperature and the useof host-tissue for final formulation and delivery technologies are novelfor this pathosystem (Valdensinia heterodoxa-Salal).

[0018] It is also significant that there is a cut-off temperature abovewhich sporulation is inhibited. Also, in the case of Valdensiniaheterodoxa, for conidia discharge, the optimum for conidia discharge atrather low temperature is unusual for the majority of fungal pathogens.More importantly, as will be apparent from the examples which follow,sporulation and discharge are severely inhibited above and below thedetermined optima.

[0019] It is also interesting that alternating light and dark treatmentsresulted in significantly lower growth rates, sporulation, and conidiadischarge. See Table 2.

[0020] Deposit Information

[0021] The above referenced Deposit was made with the InternationalDepository Authority of Canada (IDAC), 1015 Arlington Street, Winnipeg,Manitoba, R3E 3R2, Canada, under the auspices of the Budapest Treaty.The Deposit was received by the IDAC on Apr. 18, 2002, and was testedand confirmed to be viable on Apr. 22, 2002.

BRIEF DESCRIPTION OF THE DRAWING

[0022] The figure illustrates the effect of Valdensinia heterodoxa fromcolonized salal leaf pieces on intact salal plants 14 dayspost-inoculation (dpi). For each treatment, 3 g of uninoculated (controlor colonized (PFC isolate 3027) leaf pieces were placed beneath salalleaves. First disease symptoms were observed at 4 dpi.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Isolation of Valdensinia heterodoxa from Diseased Leaf Samples

[0024]Valdensinia heterodoxa (PFC 3027) having all of the identifyingcharacteristics of IDAC Deposit accession no. IDA 180402 was isolatedfrom salal leaf tissues in biologically pure form, by surfacesterilizing small pieces of infected tissue, plating them onto potatodextrose agar (PDA) or malt extract agar (MEA) and incubating in thedark at 20° C. Emerging colonies were subcultured onto PDA plates, andthen transferred to PDA slants for long-term storage at 5° C.

[0025] Starter Cultures of Valdensinia heterodoxa

[0026] Cultures of V. heterodoxa (PFC 3027) were initiated by placingsmall (V. heterodoxa) mycelium pieces from potato dextrose agar (PDA)slants maintained at 4° C. on salal PDA (SPDA; PDA amended with 40 gfresh, blended salal leaf and stem material/L dH₂O). Fungal cultureswere grown at 19/13° C. (day/night) with a 12 h day photoperiod (180-200μEm⁻²s⁻¹). Eight days after inoculation, mycelial plugs of 5-mm diameterwere transferred onto weak oatmeal agar (WOA; 15 g oatmeal agar and 12 gagar/L dH₂O) and incubated at the desired conditions, depending on theexperiment.

[0027] Effect of Temperature on Growth, Sporulation, and ConidiaDischarge of Valdensinia heterodoxa

[0028] Materials and Methods

[0029] Growth chambers were programmed at 11/6, 14/9, 17/12, and 20/15°C., respectively, with a 12 h day⁻¹ photoperiod (180-200 μEm⁻² s⁻¹).Fungal cultures of V. heterodoxa were initiated on SPDA and transferredonto WOA as described above. At 3, 6, and 9 days post-inoculation (dpi),mycelial radial growth (colony diameter), total number of conidia, andnumber of discharged conidia adhering to the lid of the Petri dish weredetermined. For each temperature treatment, five replicates were usedand the experiment was performed twice. Data were subjected to a one-wayanalysis of variance (ANOVA). In case of failed normality or equalvariance tests, a Kruskal-Wallis one-way ANOVA on ranks was usedinstead. Differences between treatment means were evaluated by aStudent-Newman-Keuls multiple comparison procedure (P=0.05).

[0030] Results

[0031] Throughout the experiment, mycelial radial growth was greatest atthe higher temperature regimes of 17/12 and 20/15° C. and was stronglyinhibited at 11/6° C. Similarly, total sporulation was improved athigher temperatures. However, conidia discharge displayed a clearoptimum at 17/12° C. and declined substantially at both temperaturesabove and below this regime. Differences in sporulation were observed asearly as 3 dpi but more pronounced towards later evaluation dates. Theresults of both trials are summarized in Table 1. TABLE 1 Effect ofday/night temperature on mycelial growth, sporulation, and conidiadischarge of Valdensinia heterodoxa (PFC 3027) at different dayspost-inoculation (dpi) Temperature Trial 1 Trail 2 (° C.)¹ 3 dpi² 6 dpi9 dpi 3 dpi 6 dpi 9 dpi Colony diameter 11/6  1.2 a  1.6 a  2.1 a  1.1 a 1.9 a  2.5 a³ (cm) 14/9  1.7 b  2.9 b  4.4 b  1.5 b  2.9 b  4.1 b 17/12 2.0 b  3.5 b  4.6 b  1.9 c  3.6 c  4.7 c 20/15  2.0 b  3.5 b  4.7 b 1.6 b  2.1 a  2.4 a Total number of 11/6  2.4 a  15.0 a  18.0 a 11.6 b 29.4 a  44.0 a conidia 14/9 10.8 ab  23.2 a 108.6 a 12.4 b  48.8 a174.0 a 17/12 23.6 b 121.2 b 318.0 b  3.0 a 206.4 b 862.4 b 20/15 26.6 b158.6 b 381.6 b  0.0 a  4.2 a  13.2 a Number of 11/6  0.0 a  0.8 a  0.8a  0.2 a³  1.6 ab³  2.4 b³ discharged 14/9  0.4 a  3.2 a  3.8 ab  0.6 a 3.4 ab  9.0 b conidia 17/12  4.2 a  4.8 a  11.6 b  0.0 a  6.8 b  72.0 c20/15  2.8 a  3.4 a  5.2 ab  0.0 a  0.0 a  0.2 a

[0032] Effect of Photoperiod on Growth, Sporulation, and ConidiaDischarge of Valdensinia heterodoxa

[0033] Materials and Methods

[0034] Three photoperiod conditions including continuous light,alternating light/dark (12 h day⁻¹ photoperiod), and continuous darknesswere investigated. Cultures of V. heterodoxa were initiated on SPDA andtransferred onto WOA as described above. Fungal cultures were grown in asingle chamber programmed at 17/12° C. with 24 h day⁻¹ light (180μEm⁻²s⁻¹). The treatment of continuous darkness was achieved by wrappingthe Petri dishes in aluminium foil. Petri dishes assigned to alternatinglight conditions were unwrapped daily at the start of the highertemperature cycle. Evaluation of mycelial growth, sporulation, andconidia discharge was performed as described above. For each photoperiodtreatment, five replicates were used and the experiment was performedtwice. Data were analyzed as for the previous experiment.

[0035] Results

[0036] Radial mycelial growth was moderately slower and totalsporulation and conidia discharge were strongly inhibited by continuousdarkness. Although all evaluated parameters were improved by acontinuous light treatment, differences between and continuous andalternating light were not always significant (Table 2). As in thetemperature experiment, differences were observed as early as 3 dpi.Sporulation data at later dates were highly variable for any of thelight treatments. Results of both trials are summarized in Table 2.TABLE 2 Effect of photoperiod duration on mycelial growth, sporulation,and conidia discharge of Valdensinia heterodoxa (PFC 3027) at differentdays post-inoculation (dpi). Trial 1 Trial 2³ Light treatment¹ 3 dpi² 6dpi 9 dpi 3 dpi 6 dpi 9 dpi Colony diameter Light  2.3 a  4.2 a  5.2 a 1.7 a  3.5 a  4.5 a (cm) Alternating  2.0 b  3.6 b  4.9 b  1.6 a  3.1ab  4.1 a Dark  2.0 b  3.4 b  4.7 b  1.6 a  2.8 b  3.7 b Total number ofLight 64.8 a 428.2 a 865.0 a 48.4 a 227.6 a 519.0 a conidia Alternating 8.8 b 137.8 b 645.2 a  6.2 b  49.4 b 168.0 b Dark  0.2 b  2.4 b  51.6 b 0.4 c  5.2 c  45.8 c Number of Light  5.4 a³  35.0 a 126.4 a  3.0 a 13.2 a  17.2 a discharged Alternating  0.6 b  5.2 b  51.0 ab  0.6 b 3.6 b  5.2 b conidia Dark  0.0 b  0.0 b  1.6 b  0.0 b  0.2 c  4.8 b

[0037] Solid-Based Formulation and Delivery Technique of Valdensiniaheterodoxa

[0038] Materials and Methods

[0039] Starter cultures of V. heterodoxa on SPDA were initiated asdescribed above. Erlenmeyer flasks containing 250 mL of liquid weakoatmeal medium (WOM; 20 g blended rolled oats/L dH₂O) were inoculatedwith 10 mycelial plugs from the starter cultures. Flasks were placed ona shaker (125 rpm) at 19/13° C. with a 12 h day⁻¹ photoperiod (180-200μEm⁻² s⁻¹).

[0040] After 7 days, resulting mycelium was harvested on double-layeredcheesecloth and blended in a Waring blender for 20 sec at high speed.Five mL of wet mycelium was added to Erlenmeyer flasks containingautoclaved salal leaf pieces (7 g litter+5 mL dH₂O). The fungal inoculumwas incubated as described above and flasks were shaken daily. After 14days, the colonized leaf material was air-dried for 2 days in a fumehood.

[0041] Salal seedlings were grown outside in 8 cm pots containingpeat-vermiculite-sand (1:1:1) and a low rate of slow release fertilizer.Plants of similar size were selected and transferred into a growthchamber at the original conditions.

[0042] Fungal inoculum was applied below salal leaves. For potted salalplants, usually several leaves protrude farther than the pot rim, hence,a structure was built beyond the rim to ensure that all leaves could bereached by discharging conidia. Plastic sheets (coroplast) were cut into14×14 cm pieces with a 6×6 cm hole in the centre. To provide a roughersurface for the leaf pieces to be placed onto, cheesecloth was cut intothe same outside dimensions as the coroplast with a ¾ slit in thecentre. Both coroplast and cheesecloth were carefully placed around theplant and fixed with a pin. For each plant, 3 g of dried leaf pieceswere applied onto the cheesecloth and watered with dH₂O from a sprayingbottle. Uninoculated leaf pieces served as the control treatment. Foreach treatment, five replicate plants were used. Pots were thentransferred to a dew chamber (100% relative humidity, 20±1° C.) for 24h; and subsequently covered with clear plastic bags and the ends tuckedunder the base of the pots. After the dew treatment, pots were movedback to the original growth chamber. Plastic bags were removed daily fora few minutes to ensure proper aeration. Leaf damage was evaluated 14dpi, based on the percentage of necrotic leaf area. Data were analyzedusing a Kruskal-Wallis one-way analysis of variance on ranks followed bya Student-Newman-Keuls multiple comparison procedure in order toevaluate differences between treatment means (P=0.05).

[0043] Results

[0044] First disease symptoms on salal plants developed 4 dpi andnecroses expanded quickly over the next days, whereas no symptoms wereobserved on the control plants (Figure). Older leaves with thickercuticle became infected as well, but necroses did not expand as fastcompared with young leaves. The origin of the necrosis was easilyidentified by the discharged conidium still attached to the leaf surfacein the centre of the necrosis. Some leaves with their upper surfacefacing down, developed disease symptoms as well. As the stomata of salalare only found on the lower leaf surface, it is assumed that conidia ofV. heterodoxa are able to penetrate their host directly. In trial 1 andtrial 2, plants exposed to the fungal inoculum developed on average 33and 39% leaf damage, respectively.

[0045] The observed leaf damage in this study is rather low comparedwith a spray application of other potential bioherbicides in whichplants are usually completely covered with fungal propagules. Althoughmost necroses developed rapidly, many leaves were not infected until theend of the experiment. On those leaves, very few conidia were found, dueto either insufficient sporulation and/or loss of discharged conidia.

[0046] The findings of this research confirm the potential of V.heterodoxa to be used as a biological control agent for salal, and inparticular, the use of colonized leaf pieces as an inoculum source anddelivery technique.

REFERENCES

[0047] D'Anjou, B 1990. Control of salal. In: Vegetation Management: AnIntegrated Approach—Proceedings of the Fourth Annual VegetationManagement Workshop (E. Hamilton, ed.). Forest Research DevelopmentAgreement Report 109: 25-26. Forestry Canada and British ColumbiaMinistry of Forests, Victoria, BC, Canada.

[0048] Haeussler, S, Coates, D, and Mather, J 1990. Autecology of commonplants in British Columbia: a literature review. Forest ResearchDevelopment Agreement Report 158: 96-102.

[0049] Lindermann, R G and Zeitoun, F 1977. Phytophthora cinnamonicausing root rot and wilt of nursery-grown native western azalea andsalal. Plant Disease Reports 61: 1045-1048.

[0050] McDonald, M A 1990. Competition for nutrients and chemicalinterference by salal (Gaultheria shallon Pursh.). In: VegetationManagement: An Integrated Approach—Proceedings of the Fourth AnnualVegetation Management Workshop (E. Hamilton, ed.). Forest ResearchDevelopment Agreement Report 109: 16-17. Forestry Canada and BritishColumbia Ministry of Forests, Victoria, BC, Canada.

[0051] Petrini, O, Stone, J, and Carroll, F E 1982. Endophytic fungi inevergreen shrubs in western Oregon: a preliminary study. CanadianJournal of Botany 60: 789-796.

[0052] Readhead, S 1974. Epistolae mycologicae IV. Valdensiniaheterodoxa Peyr. (Sclerotiniaceae). Syesis 7: 235-238.

[0053] Shamoun, S F 2000. Application of biological control tovegetation management in forestry. In: Proceedings of the TenthInternational Symposium on Biological Control of Weeds, Jul. 4-14, 1999,Montana State University, Bozeman, Mont., USA, pp. 87-96.

[0054] Shamoun, S F, Countess, R E, Vogelgsang, S, and Oleskevich, C2000. The mycobiota of salal (Gaultheria shallon) collected on VancouverIsland and the exploitation of fungal pathogens for biological control.The Canadian Phytopathological Society, Annual Meeting, Victoria, BC,Canada, Canadian Journal of Plant Pathology 22: 192 (abstract).

[0055] Wall, R E, Prasad, R, and Shamoun, S F 1992. The development andpotential role of mycoherbicides for forestry. Forestry Chronicle 68:736-741.

[0056] Watson, A K and Wall, R E 1995. Mycoherbicides: their role invegetation management in Canadian forests. In: Recent Progress in ForestBiotechnology in Canada (P. J. Charest and L. C. Duchesne, eds.).Canadian Forest Service Information Report PI-X-120: 74-82.

[0057] Voglelgsang, S., S. F. Shamoun and R. E. Countess. 2001. Inoculumproduction and biology of the fungal pathogen Valdensinia heterodoxa, apotential biological control agent for salal. Canadian Journal of PlantPathology 2001, Volume 23 (2): 208-209 (Abstract), June, 2001.

We claim:
 1. A biologically pure isolate of the naturally occurringfungus, Valdensinia heterodoxa, having all of the identifyingcharacteristics of IDAC Deposit Accession no. IDA
 180402. 2. Aherbicidal composition containing as active ingredient, the biologicallypure isolate according to claim
 1. 3. A herbicidal composition accordingto claim 2, for controlling salal (Gaultheria shallon Pursh), whereinthe active ingredient is provided as an inoculum in an agriculturallyand environmentally acceptable solid growth substrate capable ofsupporting growth of the fungus, containing a cereal grain.
 4. Acomposition according to claim 3, wherein the inoculum includes salalplant tissue, colonized by V. heterodoxa.
 5. A composition according toclaim 4, wherein the cereal grain is oats.
 6. A method for controllingsalal (Gaultheria shallon Pursh.), comprising applying to a salal plantor to a salal plant locus, an effective amount of a herbicidalcomposition containing as active ingredient, a biologically pure isolateof the fungus Valdensinia heterodoxa having all of the identifyingcharacteristics of IDAC Deposit Accession no. IDA
 180402. 7. A methodaccording to claim 6, wherein the active ingredient is provided as aninoculum in an agriculturally and environmentally acceptable solidgrowth substrate capable of supporting growth of the fungus, containinga cereal grain.
 8. A method according to claim 7, wherein the inoculumincludes salal plant tissue, colonized by H. heterodoxa.
 9. A methodaccording to claim 8, wherein the inoculum is applied to a salal plant,below leaves.
 10. A method according to claim 9, wherein the ceral grainis oats.
 11. A method for culturing V. heterodoxa, comprising (a)inoculating salal plant tissue with V. heterodoxa, and (b) growing atday/night temperatures of 20/15 to 11/6° C.
 12. A method according toclaim 11, comprising the additional step of c) incubating in agar atalternating light/dark cycles.